A new glucose microbiosensor based on glucose oxidase immobilized on the transducer surface by alternating current electrophoretic deposition (AC-EPD) is described. Deposition is carried out by applying an asymmetrical alternating current (AC) polarization between two electrodes to an enzyme dissolved in a low conductivity solution. This process, based on enzyme electrophoresis, allows the rapid manufacturing of biosensors with a very good sensitivity. Current responses to glucose up to 430 nA/mM mm(2) were obtained using a low-activity enzyme (5.6 U/mg), which is due to the preservation of the enzyme activity. The latter was confirmed by FT-IR. The thick enzymatic layer deposited on the transducer prevents a number of the interferences influencing the biosensor response, thus the need in an inner permselective membrane at formation of the biosensor recognition element is avoided. In order to optimize the linearity of the sensor response and provide a biocompatible interface in contact with biological samples, an outer layer of polyurethane (PU) was applied after the deposition of the enzyme. The resulting glucose sensor with PU outer layer had a sensitivity of at least 13 nA/mM mm(2), a linear range up to 60 mM, low interference from endogenous species, short response time (<6 s) and was stable for at least 45 days. Contrary to the GOx electrode where errors in the measurements were observed due to the moderate linearity, the sensor with the PU outer layer is shown to be effective for the determination of glucose concentrations in critically sick rabbits where the concentration of glucose might be more than 30 mM. (C) 2009 Elsevier B.V. All rights reserved.